Food analysis on microfluidic devices using ultrasensitive carbon nanotubes detectors

Microfluidic devices using carbon nanotube (CNT) materials (single-walled and two multiwalled (MWCNT)) for the analysis of selected analyte groups of significance in foods such as dietary antioxidants, water-soluble vitamins, vanilla flavors, and isoflavones involved in representative food samples have been explored for the first time. Ultrafast separations resulted in well-defined and resolved peaks with enhanced voltammetric current in comparison with those obtained from unmodified screen-printed electrodes, turning MWCNT into an ideal material for electrochemical sensing in food analysis. Resolution was improved by a factor of 2, and sensitivity was dramatically enhanced with amplification factors toward calibration slopes from 4- to 16-fold. In both qualitative and quantitative domains, this impressive performance of CNTs integrated on microfluidics allowed solving specific challenges in food environments such as the direct detection of analytes in complex natural samples and unambiguous analytes in the control of fraud, which was not possible on nonmodified surfaces, avoiding the integration of complex preconcentration steps on these microdevices. The use of these unique materials in microfluidics for food analysis has opened new expectations in "lab-on-a-chip" domains.     

Online preconcentration by transient isotachophoresis in linear polymer on a poly(methyl methacrylate) microchip for separation of human serum albumin immunoassay mixtures

Online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody by on-chip transient isotachophoresis is reported. An 800-fold signal enhancement was achieved following the preconcentration on standard cross-channel microchips made of poly (methyl methacrylate). Sample injection, preconcentration, and separation were done continuously and controlled solely by a sequential voltage switching program. The preconcentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution. The method was applied to microchip electrophoresis immunoassay of HSA. Baseline separation of HSA and its immunocomplex was achieved in 25 s in the first 1 cm of the microchannel. In a direct immunoassay, the minimum detectable concentration of fluorescent labeled HSA by laser-induced fluorescence detection was 7.5 pM.     

Strategy for on-line preconcentration in chromatographic separations

In chromatographic separations, the heights of peaks are proportional to the concentrations of sample components present in an injected mixture. In general, an increase in the peak height cannot be achieved by simply increasing the injection time or the sample plug length. An exception occurs if some form of on-line preconcentration is possible. We present a new strategy for achieving on-line preconcentration by the use of a porous chromatographic material that acts as a solid-phase extractor as well as a stationary-phase separator. We are able to realize significant on-line preconcentration using capillary columns filled with a photopolymerized sol-gel (PSG). More than 2-cm plugs of sample solution can be loaded into the capillary and concentrated using a running buffer that is the same as the injection buffer (to avoid solvent gradient effects). As a demonstration, mixtures of three different polycyclic aromatic hydrocarbons, eight different alkyl phenyl ketones, and five different peptides in solutions of aqueous acetonitrile have been injected onto the PSG column and separated by capillary electrochromatography. The preconcentration is marked in terms of peak heights, with up to 100-fold increase for the PAH mixture, 30-fold for the alkyl phenyl ketone mixture, and 20-fold for the peptide mixture. Preconcentration takes place because of the high mass-transfer rates possible in the highly porous structure, and the extent of preconcentration follows the retention factor k for a given analyte.     

Increase of reaction rate and sensitivity of low-abundance enzyme assay using micro/nanofluidic preconcentration chip

We report a novel method of increasing both the reaction rate and the sensitivity of low-abundance enzyme assay using a micro/nanofluidic preconcentration chip. The disposable preconcentration device made out of PDMS with a surface-patterned ion-selective membrane increases local enzyme/substrate concentrations for rapid monitoring of enzyme activity. As a model system, we used trypsin as the enzyme and BODIPY FL casein as the fluorogenic substrate. We demonstrated that the reaction rate of trypsin-BODIPY FL was significantly enhanced by increasing the local concentrations of both trypsin and BODIPY FL casein in the preconcentration chip. The reaction time required to turn over substrates at 1 ng/mL was only approximately 10 min compared to approximately 1 h without preconcentration, which demonstrates a significantly higher reaction rate through the increase of the concentrations of both the enzyme and substrate. Furthermore, trypsin activity can be measured down to a concentration level of 10 pg/mL, which is a approximately 100 fold enhancement in sensitivity compared to the result without the preconcentration step. This micro/nanofluidic preconcentrator chip could be used as a generic micro reaction platform to study any enzyme-substrate systems, or other biochemical reaction systems in low concentration ranges.     

Determination of lead in human saliva by combined cloud point extraction-capillary zone electrophoresis with indirect UV detection

A micelle-mediated phase separation without added chelating agents to preconcentrate trace levels of lead in human saliva as a prior step to its determination by capillary electrophoresis has been developed. The enrichment step is based on the cloud point extraction of lead with the non-ionic surfactant PONPE 7.5 in the absence of chelating agent. The surfactant-rich phase was diluted with acetonitrile and the resultant solution was injected directly into the CE instrument. Factors affecting the combined methodology such as surfactant-rich phase diluting agent, buffer pH and concentration, applied voltage, sample preparation and presence of additives were studied in detail. A BGE of 20 mM imidazole containing 30% acetonitrile, pH 6.20 was found to be optimal for the separation of lead from other saliva constituents. Indirect detection was performed at 205 nm. The detection limit value of lead for the preconcentration of 8 ml of saliva was 11.4 microg l(-1). The calibration graph using the preconcentration system was linear with a correlation coefficient of 0.997 at levels near the detection limits up to at least 400 microg l(-1). The reproducibility (R.S.D.) on the basis of migration time and peak area were better than 0.68 and 3.6%, respectively. The method was successfully applied to the determination of lead in human saliva.     

Determination of Nd isotopes in water: a chemical separation technique for extracting Nd from seawater using a chelating resin

A new preconcentration technique for the determination of the concentration and isotopic composition of neodymium in aqueous samples is presented. The method uses a resin, Nobias PA1 from Hitachi High-Technologies, which has a hydrophilic methacrylate polymer backbone where the functional groups ethylenediaminetriacetic and iminodiacetic acids are immobilized. The function of the resin has been tested by preconcentrating 110-350 pmol of Nd from test solutions as well as from natural brackish water and seawater samples with different salinities and Nd concentrations. Samples were loaded onto the resin after the pH was adjusted, and the Nd fraction was eluted using 3 M HNO(3). The method shows yields of about 90% or higher at pH 6 when the samples were buffered using ammonium acetate. Without the addition of buffer the yield decreased to below 80%. The isotopic composition of Nd in samples preconcentrated using Nobias PA1 agree within error with published data or data obtained by other methods. The total blank, including contributions from preconcentration, separation, and mass spectrometry, is estimated to be 0.2-0.4 pmol (30-60 pg) of Nd. The described preconcentration method, which can be used in the field, is easy, fast (about 8 h for a 3.6 kg sample), and reliable for preconcentration of Nd from a seawater matrix.     

Determination of 16 phthalate metabolites in urine using automated sample preparation and on-line preconcentration/high-performance liquid chromatography/tandem mass spectrometry

We developed an on-line solid-phase extraction (SPE) method, coupled with isotope dilution high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) and with automated sample preparation, to simultaneously quantify 16 phthalate metabolites in human urine. The method requires a silica-based monolithic column for the initial preconcentration of the phthalate metabolites from the urine and a silica-based conventional analytical column for the chromatographic separation of the analytes of interest. It uses small amounts of urine (100 microL), is sensitive (limits of detection range from 0.11 to 0.90 ng/mL), accurate (spiked recoveries are approximately 100%), and precise (the inter- and intraday coefficients of variation are <10%). The method is not labor intensive, and, because pretreatment of the urine samples was performed automatically using an HPLC autosampler, involves minimal sample handling, thus minimizing exposure to hazardous chemicals. The method was validated on spiked, pooled urine samples and on urine samples from 43 adults with no known exposure to phthalates. The high sensitivity and high throughput (HPLC run time, including the preconcentration step, is 27 min) of this analytical method combined with the ease of use and effective automated sample preparation procedure make it suitable for large epidemiological studies to evaluate the prevalence of human exposure to phthalates.     

Optimization of the preconcentration system of cadmium with 1(2-thiazolylazo)-p-cresol using a knotted reactor and flame atomic absorption spectrometric detection

The present paper proposes an on-line preconcentration procedure for cadmium determination in drinking water samples. It is based on the precipitation of cadmium(II) ions on a knotted reactor (KR) using 1(2-thiazolylazo)-p-cresol (TAC) as complexing reagent. The optimization step was performed using a full factorial design involving the variables: pH, eluent concentration (nitric acid) and TAC concentration. The results of this experiment demonstrated that these variables at chosen levels are not statistically significant. Under optimized experimental established conditions, analytical parameters for the preconcentration method were: a detection limit of 40.0 ng/l, precision as relative standard deviation (RSD) of 1.2 and 1.0%, for cadmium concentration of 2.5 and 20.0 microg/l, respectively. The preconcentration factor considering the slopes of the analytical curves with and without preconcentration is 23 for a sample volume of 10 ml. This system shows a sampling frequency of 25 h(-1). In order to check the accuracy, the standard reference material, NIST SRM 1643d trace elements in water was analyzed. A comparison, using t-test demonstrates that there is not significant difference among the achieved results with proposed method and the certified values. The addition/recovery experiments in the samples analyzed demonstrated the accuracy and applicability of the system developed for cadmium determination in water samples.     

Integrated preconcentration SDS-PAGE of proteins in microchips using photopatterned cross-linked polyacrylamide gels

The potential of integration of functions in microfluidic chips is demonstrated by implementing on-chip preconcentration of proteins prior to on-chip protein sizing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two polymeric elements-a thin (approximately 50 microm) size exclusion membrane for preconcentration and a longer (approximately cm) porous monolith for protein sizing-were fabricated in situ using photopolymerization. Contiguous placement of the two polymeric elements in the channels of a microchip enabled simple and zero dead volume integration of the preconcentration with SDS-PAGE. The size exclusion membrane was polymerized in the injection channel using a shaped laser beam, and the sizing monolith was cast by photolithography using a mask and UV lamp. Proteins injected electrophoretically were trapped on the upstream side of the size exclusion membrane (MW cutoff approximately 10 kDa) and eluted off the membrane by reversing the electric field. Subsequently, the concentrated proteins were separated in a cross-linked polyacrylamide monolith that was patterned contiguous to the size exclusion membrane. The extent of protein preconcentration is easily tuned by varying the voltage during injection or by controlling the sample volume loaded. Electric fields applied across the nanoporous membrane resulted in a concentration polarization effect evidenced by decreasing current over time and irreproducible migration of proteins during sizing. To minimize the concentration polarization effect, sieving gels were polymerized only on the separation side of the membrane, and an alternate electrical current path was employed, bypassing the membrane, for most of the elution and separation steps. Electrophoretically sweeping a fixed sample volume against the membrane yields preconcentration factors that are independent of protein mobility. The volume sweeping method also avoids biased protein loading from concentration polarization and sample matrix variations. Mobilities of the concentrated proteins were log-linear with respect to molecular weight, demonstrating the suitability of this approach for protein sizing. Proteins were concentrated rapidly (<5 min) over 1000-fold followed by high-resolution separation in the sieving monolith. Proteins with concentrations as low as 50 fM were detectable with 30 min of preconcentration time. The integrated preconcentration-sizing approach facilitates analysis of low-abundant proteins that cannot be otherwise detected. Moreover, the integrated preconcentration-analysis approach employing in situ formation of photopatterned polymeric elements provides a generic, inexpensive, and versatile method to integrate functions at chip level and can be extended to lowering of detection limits for other applications such as DNA analysis and clinical diagnostics.     

Simultaneous determination of bromide and iodide as acetone derivatives by gas chromatography and electron capture detection in natural waters and biological fluids

Oxidation of bromide and iodide ions in acidic solutions in the presence of acetone forms the corresponding acetone derivatives. Iodate was reduced with thiosulfate prior to the determination. After extraction with benzene the bromo- and iodoacetone were measured by gas chromatography using electron capture detection. The bromide and iodide contents of rainwater, drinking water, river water, seawater, oil brine, common salt, cow milk, and human blood serum were determined. The relative standard deviations for bromide at 10(-7) M and for iodide at 10(-8) M concentration were 1.9% and 3.0%, respectively, using a 10-mL sample for the determination without preconcentration.     

Fluidic preconcentrator device for capillary electrophoresis of proteins

A new preconcentration device was developed for analysis of proteins by capillary electrophoresis (CE). The microfluidic device uses an electric field to capture proteins that pass through the system. The capture zone is maintained in the flow stream by the interaction between hydrodynamic and electrical forces. The device consists of a flow channel made of PEEK tubing with two electrical junctions, each of which is covered with a conductive membrane. A syringe pump provides the flow stream and also allows the injection of up to 13.5 microL of a dilute sample. The system can be easily connected to a CE device postcapture for off-line preconcentration of proteins. For the proteins used in this study, preconcentration factors up to 40-fold can be achieved. CE detection limits for bovine carbonic anhydrase, alpha-lactalbumin and beta-lactoglobulins A and B were in the nanomolar range using UV detection at 200 nm. Preconcentration is dependent on both time and initial protein concentration. We show the possibility of using an off-line fluidic preconcentrator device employing counterflow capillary electrophoresis with minimum sample manipulation, achieving detection limits similar to on-line approaches.     

Ultra-trace determination of silver in water samples by electrothermal atomic absorption spectrometry after preconcentration with a ligand-less cloud point extraction methodology

A very simple and ligand-less cloud point extraction (CPE) methodology for the preconcentration of ultra-trace amounts of silver as a prior step to its determination by electrothermal atomic absorption spectrometry (ETAAS) has been developed. The method is based on the extraction of silver at pH 9 by using non-ionic surfactant polyethyleneglycolmono-p-nonylphenylether (PONPE 7.5) without adding any chelating agent. Several important variables that affect the CPE efficiency and ETAAS signal were investigated and optimized. The preconcentration of 15 ml sample solution allowed us to achieve an enhancement factor of 60. The calibration graph using the preconcentration system was linear in the range of 5-100 ngl(-1) with a correlation coefficient of 0.9991. The lower limit of detection (3s) obtained in the optimal conditions was 1.2 ngl(-1). The relative standard deviation (R.S.D.) for eight replicate determinations at 30 ngl(-1) Ag level was 4.2%. The proposed method was successfully applied to the ultra-trace determination of silver in water samples.     

Genotoxicity screening using biocatalyst/DNA films and capillary LC-MS/MS

Detection of DNA adducts can serve as a basis for genotoxicty screening of new chemicals and drugs. We report here a simple, sensitive procedure for this purpose using films containing DNA and a biocatalyst to mimic the metabolic action of human liver cytochrome P450s. DNA adducts formed from an in-situ-generated toxic metabolite (styrene oxide) were detected at subpicomole levels after neutral thermal hydrolysis of the DNA films and analysis with capillary liquid chromatography with on-line column preconcentration and MS/MS detection. An on-line column switching system allowed for increased sample loading volume and analyte preconcentration. This approach provides an estimate of the relative rate of DNA damage.     

A flow injection on-line multiplexed sorption preconcentration procedure coupled with flame atomic absorption spectrometry for determination of trace lead in water, tea, and herb medicines

One of the limitations in previous flow injection (FI) sorption preconcentration procedures in a knotted reactor (KR), which have been carried out exclusively with a single continuous sample injection over a certain period, is the relatively low retention efficiency (typically 40-50%). Although the sensitivity of such systems could be improved by properly increasing sample preconcentration time, sample loading flow rate, or both, further improvement of the sensitivity has been limited by the narrow linearity of the relationship between signal intensity and preconcentration time or sample loading time. In this work, a novel on-line FI multiplexed sorption preconcentration procedure with repetitive sample injections was developed to overcome the above problems in the previous systems. In contrast to previous FI preconcentration systems, the proposed multiplexed preconcentration procedure evenly divides a single longer sample injection step into several shorter substeps while the total preconcentration time is still kept constant. To demonstrate its merits, the proposed FI on-line KR multiplexed sorption preconcentration system was combined with flame atomic absorption spectrometry (FAAS) for determination of trace lead in water, tea, and herb medicines. The lead in the sample solution on-line reacted with ammonium pyrrolidine dithiocarbamate, and the resultant analyte complex was sorbed on the inner walls of the KR. The residual sample solution was then removed from the KR with an air flow. The above two steps were repeated eight times with a total preconcentration time of 120 s. The sorbed analyte was eluted from the KR with 4.5 mol L(-1) HCl for on-line FAAS detection. The present multiplexed preconcentration procedure with eight repetitive sample injections for a total preconcentration time of 120 s gave a retention efficiency of 92%, twice that obtained by one single sample injection preconcentration (47%). In addition, the linear ranges of the diagrams of absorbance against sample loading flow rate and sample loading time were extended, offering more potential for achieving high sensitivity by increasing sample loading rates or sample loading time compared to the previous one single continuous sample injection preconcentration procedure. At a sample loading flow rate of 3.6 mL min(-1) for a total preconcentration period of 120 s, an enhancement factor of 57 and a detection limit (3sigma) of 8 microg L(-1) were obtained. The precision was 1.4% (RSD, n = 11) at the 200 microg L(-1) level. The developed method was successfully applied to the determination of trace lead in various water samples, herb medicines, and a certified tea reference material.     

Preconcentration of uranium on POLIORGS 34-n fibrous filled sorbent from natural waters

Sorption preconcentration of U(VI) on POLIORGS 34-n fibrous filled sorbent with amidoxime groups from model solutions like natural water was studied under static and dynamic conditions. The equilibrium (distribution coefficient) and kinetic (diffusion coefficient) parameters of the sorption were determined. Parameters of dynamic preconcentration of uranium by sorption on disks and in a column packed with POLIORGS 34-n under indicated conditions were calculated by mathematical modeling using the experimental data.     

Signal enhancement in HPLC/microcoil NMR using automated column trapping

A new HPLC NMR system is described that performs analytical separation, preconcentration, and NMR spectroscopy in rapid succession. The central component of our method is the online preconcentration sequence that improves the match between postcolumn analyte peak volume and microcoil NMR detection volume. Separated samples are collected on to a C18 guard column with a mobile phase composed of 90% D2O/10% acetonitrile-D3 and back-flushed to the NMR microcoil probe with 90% acetonitrile-D3/10% D2O. To assess the performance of our unit, we separated a standard mixture of 1 mM ibuprofen, naproxen, and phenylbutazone using a commercially available C18 analytical column. The S/N measurements from the NMR acquisitions indicated that we achieved signal enhancement factors up to 10.4 (+/-1.2)-fold. Furthermore, we observed that preconcentration factors increased as the injected amount of analyte decreased. The highest concentration enrichment of 14.7 (+/-2.2)-fold was attained injecting 100 microL of solution of 0.2 mM (approximately 4 microg) ibuprofen.     

On-column ion-exchange preconcentration of inorganic anions in open tubular capillary electrochromatography with elution using transient-isotachophoretic gradients. 3. Implementation and method development

A solid-phase extraction method based on an ion-exchange retention mechanism has been used for in-line preconcentration of inorganic anions prior to their separation by capillary electrophoresis (CE). A single capillary containing a preconcentration and a separation zone has been used in a commercial CE instrument without instrumental modification. Analyte anions were retained on a preconcentration zone comprising an adsorbed layer of cationic latex particles, while separation was achieved in a separation zone comprising fused silica modified by adsorption of a cationic polymer. Elution of the adsorbed analytes was achieved using an eluotropic gradient formed by a transient isotachophoretic boundary between a fluoride electrolyte and a naphthalenedisulfonate electrolyte. Optimization of the electrolyte concentrations, sample injection times, and back-flushing times allowed the successful separation of sub-ppb levels of inorganic anions using a 100-min injection at 2 bar pressure, introducing over 40 capillary volumes of sample. A method based on a 10-min injection allowed a 100-fold increase in sensitivity over conventional hydrodynamic injection for Br-, I-, NO3-, CrO4(2-), and MoO4(2-) with a total analysis time of 25 min. Detection limits were dependent on the injection time but were in the range 2.2-11.6 ppb for a 10-min injection time. This approach was used to determine NO3- in Antarctic ice cores where the analysis could be performed using a sample volume 100 times less than that used for ion chromatography.     

Direct analysis of trace phenolics with a microchip: in-channel sample preconcentration, separation, and electrochemical detection

A micrototal analytical method assembling in-channel preconcentration, separation, and electrochemical detection steps has been developed for trace phenolic compounds. A micellar electrokinetic chromatography separation technique was coupled with two preconcentration steps of field-amplified sample stacking (FASS) and field-amplified sample injection (FASI). An amperometric detection method with a cellulose-dsDNA-modified, screen-printed carbon electrode was applied to detect preconcentrated and separated species at the end of the channel. The microchip was composed of three parallel channels: first, two are for the sample preconcentration using FASS and FASI methods, and the third one is for the separation and electrochemical detection. The modification of the electrode surface improved the detection performance by enhancing the signal-to-noise characteristic without surface fouling of the electrode. The method was examined for the analysis of eight phenolic compounds. Experimental parameters affecting the analytical performance of the method were assessed and optimized. The preconcentration factor was increased by about 5200-fold as compared with a simple capillary zone electrophoretic analysis using the same channel. Reproducible response was observed during multiple injections of samples with a RSD of <8.0%. The calibration plots were shown to be linear (with the correlation coefficient between 0.9913 and 0.9982) over the range of 0.4-600 nM. The sensitivity was between 0.17 +/- 0.001 and 0.48 +/- 0.006 nA/nM, with the detection limit of approximately 100 to approximately 150 pM based on S/N = 3. The applicability of the method to the direct analysis of trace phenolic compounds in water samples was successfully demonstrated.     

Solid-phase extraction method for preconcentration of trace amounts of some metal ions in environmental samples using silica gel modified by 2,4,6-trimorpholino-1,3,5-triazin

A method was proposed for the preconcentration of some transition elements at trace levels using a column packed with silica gel modified by a synthetic ligand. Metal ions were adsorbed on 2,4,6-trimorpholino-1,3,5-triazin modified silica gel, then analytes retained on the adsorbent were eluted by 1 mol L⁻¹ hydrochloric acid and determined by flame atomic absorption spectrometry (FAAS). The influences of some experimental parameters including pH of the sample solution, weight of adsorbent, type, concentration and volume of eluent, flow rates of the sample solution and eluent, and sample volume on the preconcentration efficiency have been investigated. The influences of some matrix elements were also examined. The method also was used for simultaneous preconcentration of these elements and the method was successfully applied to the preconcentration and determination of them. The detection limits of the method for Ni²⁺, Co²⁺, Cd²⁺ and Zn²⁺were 0.29, 0.20, 0.23 and, 0.30 ng mL⁻¹, respectively. The application of this modified silica gel to preconcentration of investigated cation from tap water, lake water, urine and apple leaves gave high accuracy and precision (relative standard deviation (R.S.D.) <3%).     

Liquid-phase microextraction as an on-line preconcentration method in capillary electrophoresis

A simple and efficient sample preconcentration method for capillary electrophoresis has been developed using liquid-phase microextraction (LPME). A thin layer of an organic liquid was used to separate a drop of the aqueous acceptor phase hanging at the inlet of a capillary from the bulk aqueous donor phase. The donor-phase pH was 1.0, and the acceptor phase pH was 9.5. This pH difference caused the preconcentration of the acidic compounds, fluorescein and fluorescein isothiocyanate, into the acceptor-phase drop. Enrichment factors of 3 orders of magnitude were obtained with 30-min LPME at 35 degrees C.